Endoglycosidase S (EndoS) is secreted by a number of serotypes of Streptococcus pyogenes and has a specific endoglycosidase activity on native IgG by hydrolyzing the conserved glycans attached to the asparagine 297 residue on the heavy chains of IgG, Collin and Olsen, The EMBO Journal, 2001, 20 3046-3055. EndoS is the first known bacterial enzyme with a unique specificity for native IgG. In contrast, the activities of other known endoglycosidases require or are enhanced by denaturation of the glycoprotein substrate.
Antibodies such as IgG have many applications in basic research as well as in diagnostics and drug development. In some of these applications, such as immunohistochemistry, immunoassays, tumour detection, radiotherapy, crystallographic studies of antibody binding sites and immunotargeting, it is more convenient to use Fab fragments than whole IgG molecules. Some of the advantages of using Fab fragments are that they will not be affected by Fc receptors on cells or precipitate antigen, they display a reduced immunogenicity and are less susceptible to phagocytosis, and that radiolabelled Fab fragments are more rapidly cleared from tissue than whole IgG molecules. For other applications, it is desirable to use Fc fragments of IgG. In further applications, it may be desirable to use deglycosylated versions of the antibodies or other glycoproteins.
The cleavage of IgG into Fab and Fc fragments is most often carried out using proteolytic enzymes such as pepsin or papain. These enzymes often cleave other proteins, so the cleavage reaction generally has to be performed on a purified IgG fraction. Furthermore, pepsin and papain typically cleave IgG in more than one place. This means that the fragments obtained often do not correspond to whole Fab or Fc fragments, and even if cleavage does result in Fab and Fc fragments, they are typically susceptible to further cleavage into smaller fragments. The isolation of Fc fragments from Fab fragments is most often carried out using protein A or G affinity separation columns, which utilise the Fc-binding properties of the bacterial proteins A and G.
Many different glycoproteins have utility in therapeutic applications. Methods to analyse the glycosylation of such proteins have utility in the research and development of the proteins as therapeutics. It may also be desirable to provide deglycosylated versions of these proteins.